Reciprocal Connections between Parvalbumin-Expressing Cells and Adjacent Pyramidal Cells Are Regulated by Clustered Protocadherin γ

Abstract Functional neural circuits in the cerebral cortex are established through specific neural connections between excitatory and various inhibitory cell types. However, the molecular mechanisms underlying synaptic partner recognition remain unclear. In this study, we examined the impact of clustered protocadherin-γ (cPcdhγ) gene deletion in parvalbumin-positive (PV+) cells on intralaminar and translaminar neural circuits formed between PV+ and pyramidal (Pyr) cells in the primary visual cortex (V1) of male and female mice. First, we used whole-cell recordings and laser-scan photostimulation with caged glutamate to map excitatory inputs from layer 2/3 to layer 6. We found that cPcdhγ-deficient PV+ cells in layer 2/3 received normal translaminar inputs from Pyr cells through layers 2/3–6. Second, to further elucidate the effect on PV+-Pyr microcircuits within intralaminar layer 2/3, we conducted multiple whole-cell recordings. While the overall connection probability of PV+-Pyr cells remained largely unchanged, the connectivity of PV+-Pyr was significantly different between control and PV+-specific cPcdhγ-conditional knock-out (PV-cKO) mice. In control mice, the number of reciprocally connected PV+ cells was significantly higher than PV+ cells connected one way to Pyr cells, a difference that was not significant in PV-cKO mice. Interestingly, the proportion of highly reciprocally connected PV+ cells to Pyr cells with large unitary IPSC (uIPSC) amplitudes was reduced in PV-cKO mice. Conversely, the proportion of middle reciprocally connected PV+ cells to Pyr cells with large uIPSC amplitudes increased compared with control mice. This study demonstrated that cPcdhγ in PV+ cells modulates their reciprocity with Pyr cells in the cortex.


Introduction
GABAergic interneurons in the cortex are classified into numerous types.Parvalbumin-positive (PV 1 ) cells are a major inhibitory neuron type (Celio, 1986;Gonchar and Burkhalter, 1997;Kawaguchi and Kubota, 1997).Unlike other inhibitory neuronal cell types, PV 1 cells exhibit a characteristic fast-spiking firing pattern (Kawaguchi, 1995) and synapse with proximal dendrites and cell bodies of their target excitatory neurons (Kawaguchi, 1995;Tamás et al., 1997).This configuration is believed to provide robust inhibition to target cells and regulate their firing timing (Cardin, 2018).Additionally, PV 1 cells participate in regulating responses to visual stimuli (Atallah et al., 2012) and formation of g waves (Sohal et al., 2009), which are associated with information processing (Singer, 1993).PV 1 cells have inputoutput relationships with specific layers and neurons depending on the cell type.There is also selectivity in the binding relationships with neighboring excitatory and inhibitory cells, forming nonrandom microcircuits (Yoshimura and Callaway, 2005;Morishima et al., 2017), while PV 1 cells target adjacent pyramidal (Pyr) cells nonspecifically (Packer and Yuste, 2011).However, the molecular mechanisms underlying the connection specificity between excitatory and inhibitory neurons remains unclear.
Previous studies have indicated that cell lineage plays a crucial role in establishing specific connections among excitatory cells.Excitatory cells originating from the same single radial glial cell preferentially form synaptic contacts in the sensory cortex (Packer and Yuste, 2011).Our previous findings demonstrated that clonal layer four excitatory cells in the barrel cortex preferentially form reciprocal connections compared with nonclonal layer four excitatory cells.Moreover, cell lineage-dependent reciprocal connections are significantly reduced on deletion of cPcdh (Tarusawa et al., 2016).Lv et al., also reported that patterned cPcdh expression in individual cells regulates the connectivity of clonal excitatory neurons in the cortex (Lv et al., 2022), further indicating that cPcdh is one of the potential molecules involved in intercellular target recognition.Cortical GABAergic interneurons originate from a different cell lineage compared with glutamatergic neurons (Marín and Müller, 2014).Deletion of cPcdhg in cortical inhibitory cells is accompanied by cell death during early postnatal stages (Carriere et al., 2020;Leon et al., 2020), complicating our understanding of the specific function of cPcdhg in neural circuit formation within cortical inhibitory neurons.
Deletion of cPcdhab g and cPcdhg in mice results in deficits in left-right alternation of locomotor-like activity in spinal cords, while cPcdhab g knock-out (KO) hippocampal dissociated cultures exhibit abnormally correlated neural activity (Hasegawa et al., 2016(Hasegawa et al., , 2017)), underscoring the role of cPcdhs in neural circuit formation.
In this study, we used PV-Cre mice to delete cPcdhg in parvalbumin-positive (PV 1 ) cells because PV expression starts around postnatal day 14 (P14) in visual cortex (Lecea et al., 1995), which is already past the peak of programmed interneuronal cell death (Wong et al., 2018).The PV 1 cells lacking cPcdhg normally survived at postnatal day 21 by avoiding early neonatal cell death.We show that the deletion of cPcdhg in PV 1 cells changes the unique connectivity of each PV 1 cell with adjacent Pyr cells.These results suggest that cPcdhs regulate microcircuit formation between the excitatory and inhibitory cells.

Animals
Animal experiments in this study adhered to the Osaka University Experimental Regulations (approval number: FBS-14-002-1).Mice were kept in a 24-h cycle with 12 h of light and 12 h of darkness.
Brains from P0 mice of each genotype were homogenized in five volumes of H buffer [20 mM Tris-HCl (pH 8.0), 2 mM EDTA (pH 8.0), 0.32 M sucrose] supplemented with cOmplete protease inhibitor (Roche, catalog #04693116001).After centrifugation at 20,000 Â g for 1 h at 4°C, the pellet (P2 fraction) was solubilized with S buffer [20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1.3% Triton X-100] with cOmplete protease inhibitor for 1 h at 4°C.After centrifugation at 20 000 Â g for 1 h at 4°C, the supernatant was collected, and the protein concentration was determined by BCA protein assay (Thermo Fisher Scientific, catalog #23227).We used 40 mg of proteins for SDS-PAGE and blotted them onto a nitrocellulose membrane, followed by the standard procedure.The membranes were blocked with 2% nonfat dry milk in TBS-T (200 mM NaCl, 40 mM Tris, 0.1% Tween 20), then incubated with rabbit anti-pan-cPcdhg and rabbit anti-b-actin (13E5, Cell Signaling Technology, catalog #4970S) overnight at 4°C.The next day, the membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibody for 1 h at room temperature.Membranes were then washed and developed with GE HealthcareECL prime (Cytiva, catalog #RPN2232).All images were acquired and analyzed using the ChemiDoc MP Imaging System (Bio-Rad).
P21 mice were anesthetized with isoflurane and subsequently decapitated, and their brains were quickly frozen in dry-ice-cooled hexane, before being stored at À80°C.The primary visual cortex (V1) was sectioned to a thickness of 10 mm and RNAs were detected by ISHpalette (Nepagene) using the manufacturer's protocol.Twenty pairs of split-initiator DNA probes targeting the Pcdhg constant region and nine pairs of split-initiator DNA probes for PV were designed (Table 1).The probes were mixed for hybridization at a concentration of 20 nM in an HCR hybridization buffer and incubated at 37°C overnight.HCR amplification occurred using ISHpalette Short hairpin amplifier, SaraFluor 488-S45 for Pcdhg, and ISHpalette Short hairpin amplifier, ATTO647N-A161 for PV at 25°C for 2 h.

Cell counting of PV 1 cells in the visual cortex
Mice were anaesthetized with isoflurane, perfused transcardially first with 25 mM PBS and then with 4% paraformaldehyde (PFA) in PB (pH 7.3).The brains, removed from the skull, were stored in PFA at 4°C overnight, then transferred to 25 mM PBS with 30% sucrose and kept at 4°C overnight.Cryostat sections were cut at 20-mm thickness, and slides were washed three times in 25 mM PBS for 5 min.For immunofluorescence, the slides were incubated with DAPI (1:20,000) for 20 min.They were then washed in 25 mM PBS for 5 min.Images were taken at 10Â magnification using a KEYENCE BZ-9000 microscope.Cortical layers were identified based on their distinct cell densities.Each layer was enclosed in a 200mm-wide square based on DAPI, and the number of cells within each layer was counted manually.Five fields of view were used per mouse for the analysis.

Whole-cell recordings
All recordings were performed in the V1 monocular region.Fluorescent protein-expressing and-nonexpressing cells were identified under fluorescent and infrared differential interference contrast optics with an 40Â, 0.8 NA water immersion lens (BX-50WI, Olympus).Normal ACSF oxygenated with a CO 2 /O 2 gas mixture was flowed into the submerged chamber of the upright microscope.Glass patch pipettes (BF150-110-7.5,Sutter Instrument; 5-7 MV) were filled with a solution containing 130 mM K-gluconate, 8 mM KCl, 1 mM MgCl 2 , 0.6 mM EGTA, 10 mM HEPES, 3 mM MgATP, 0.5 mM Na 2 GTP, 10 mM Na-phosphocreatine, and 0.2% biocytin (pH 7.3 adjusted with KOH) for PV 1 cells and for action potential recordings of Pyr cells; and 130 mM Cs-gluconate, 8 mM CsCl, 1 mM MgCl 2 , 0.6 mM EGTA, 3 mM MgATP, 0.5 mM Na 2 GTP, 10 mM HEPES, 10 mM Na-phosphocreatine, and 0.2% biocytin (pH 7.3 adjusted with CsOH) for uIPSC recordings in Pyr cells.Neurons with the soma located at least 50 mm below the cut surface of the slice were recorded.For the analysis, we selected cells with a series resistance of ,25 MV.We did not use series resistance compensation.All recordings were conducted using a MultiClamp 700B (Molecular Devices) amplifier, and data were analyzed using pClamp11 software (Molecular Devices).PV 1 cells were identified using tdTomato fluorescence.Pyr cells were identified by their triangle-like shape.After recording, the slices were fixed in 4% PFA in 0.1 M PB overnight at 4°C.After fixation, the recorded cells were visualized by staining with Alexa Fluor 488-conjugated streptavidin (code: 016-540-084, Jackson ImmunoResearch) to confirm the cell type and location.Upon rupture of the cell membrane, the resting membrane potential was immediately recorded.The firing pattern evoked by depolarizing current injections, was measured in the current-clamp mode.The input resistance was recorded by applying a 5-mV square wave pulse in the voltage-clamp mode.In the PV 1 cells, the firing characteristics were analyzed using the first action potential generated by the depolarizing current injection.For the simultaneous whole-cell recording of PV 1 cell-Pyr cell pairs, neurons located within 50 mm or 60-100 mm range were targeted.In all double or triple recordings, synaptic connections between neurons were assessed in bidirectionally by applying brief (2 ms) depolarizing voltage pulses (minimum 50 trials) to evoke action potentials in one cell, while recording synaptic responses in the other.The holding membrane potential of PV 1 cells was set to -70 mV for uEPSCs recordings, and Pyr cells were set to 0 mV for uIPSCs recordings.The holding potentials were corrected for the liquid junction potential.To determine the paired pulse ratio, action potentials were induced twice at 50-ms intervals for uEPSCs and 100-ms intervals for uIPSCs.

Laser photostimulation
The experiment was performed as previously described (Yoshimura and Callaway, 2005;Ishikawa et al., 2014).Photostimulation was achieved through focal photolysis of RuBi-caged glutamate (Tocris; 3574) using 10-ms flashes of blue light (440 nm) emitted by a diode laser (FV5-LDPSU, Olympus).The light was focused onto the slices using a 4Â, 0.16 NA microscope objective.Laser power was adjusted to induce action potential in the recorded cell at one or two photostimulation spots in cell-attached mode.Photostimulation-evoked EPSCs were recorded in the layer 2/3 PV 1 cells.Typically, photostimulations were applied to 9 Â 22 spots surrounding the recorded cell at 4.5-s intervals in a quasi-random sequence.To demonstrate that the direct responses and uEPSCs could be distinguished, we recorded 9 Â 20 uEPSCs of PV 1 cells through photostimulation and introduced 1 mM tetrodotoxin (TTX) to block Na 1 channels.The same conditions were repeated for recording 10 min later (Dantzker and Callaway, 2000).

Analysis for electrophysiology
The kinetics of action potentials, unitary EPSCs (uEPSCs), and unitary IPSCs (uIPSCs) were analyzed using Clampfit 11 (Molecular Devices).The action potential threshold was defined as the point where the membrane potential change rate surpassed 10 mV/ms.The amplitude measurements of uEPSCs and uIPSCs included failure events, but such events were excluded from the kinetic analysis of uEPSCs and uIPSCs.EPSCs induced by photostimulation were analyzed as described earlier (Yoshimura et al., 2005).The maps of photostimulation sites were aligned with laminar borders in fixed and stained tissues, and each site was assigned a laminar identity.The records from stimulus sites on the layer boundaries were analyzed as records of the layer where the stimulus site's center point belonged.Electrical recordings obtained from the photostimulation were analyzed using MiniAnalysis (Synaptosoft) and other custom software written in MATLAB (RRID:SCR_001622).We measured peak time and amplitude of all EPSCs occurring 10-ms postphotostimulation for 120 ms.The count of EPSCs evoked by photostimulation included temporally overlapped EPSCs and isolated ones.

Dendritic morphologic analysis
The dendritic morphology of biocytin-filled PV 1 cells in layer 2/3, which were visualized by streptavidin staining, was captured using a confocal microscope (SpinSR, Olympus) with a x40 lens.The thickness of the Z slices was set at 1-mm intervals, ranging from the slice surface to the point where the fibers were not visible (;100 mm).Dendritic morphology was traced with the simple neurite tracer (SNT) plugin on ImageJ/FIJI (https://imagej.net/software/fiji/), and Sholl analysis was conducted using the SNT plugin.Concentric circles were positioned in 10-pixel (5.88 mm) steps from the cell body center.

Statistical analysis
Statistical analysis was performed using the Mann-Whitney U test, Welch's t test or two-way ANOVA when two groups were compared.Dunn's multiple comparisons test, Bonferroni's multiple comparisons test and Holm-Sídák's multiple comparisons test were also performed when more than two groups were compared.x 2 and Fisher exact tests were also performed for group comparison.A p-value of ,0.05 was considered statistically significant.

Results
Normal development of cPcdhc fl/fl; PV-Cre mice To investigate whether cPcdhg in PV 1 cells contributes to forming synaptic connections, cPcdhg was specifically deleted in PV 1 cells.Previous reports have shown that cPcdhg conditional KO mice crossed with gad2-Cre, Nkx2.1-Cre, and SST-Cre mice display extensive inhibitory neural apoptosis in the cortex (Carriere et al., 2020;Leon et al., 2020), making it challenging to investigate the functional role of cPcdhg in neural circuit formation.We also used cPcdhg flox mice in which g CR1 exon of cPcdhg was floxed (Hoshino et al., 2023).To examine cPcdhg deficiency by lacking g CR1 exon, we produced the g CR1 exon lacking allele by using Cre-induced mitotic recombination of Sycp-Cre transgenic mice (Fig. 1A,C).Homozygous pups were born but exhibited irregular breathing, repeated limb tremors, and died within 1 d of birth (Fig. 1B), similar phenotypes to previous reported cPcdhg KO mice (Wang et al., 2002).And we also confirmed complete deletion of cPcdhg proteins in the g CR1 exon lacking homozygote brains (Fig. 1D).To produce conditional cPcdhg lacking mice specifically in PV 1 cells, we crossed between cPcdhg flox and PV-Cre mice.PV expression starts around P14 in visual cortex, so that the Cre-induced cPcdhg deletion of PV 1 cells in cPcdhg fl/fl; PV-Cre; Ai14/Ai14 or Ai14/1 (PV-cKO) mice occur after this age.Therefore the PV 1 cells in PV-cKO mice avoided cell death during early neonatal stages (,P14) in the cortex (Carriere et al., 2020;Leon et al., 2020).To confirm the expression and deletion of cPcdhg on P21, in situ hybridization for cPcdhg mRNA detection was performed (Fig. 2A-C).The mRNA of cPcdhg was detected in excitatory and PV 1 cells in cPcdhg 1/1; PV-Cre; Ai14/Ai14 or Ai14/1 mice (control mice), whereas the signals for cPcdhg undetectable only in the PV 1 cells of cPcdhg fl/fl; PV-Cre; Ai14/Ai14 or Ai14/1 mice (PV-cKO mice; Fig. 2A-C).Using a smFISH method with double PV and cPcdhg probes, we also confirmed the PV 1 cells specific cPcdhg mRNA deletion in the primary visual cortex at P21 (Fig. 2D).Similar cell number and  distribution of PV 1 cells in the visual cortex were observed between control and PV-cKO mice at P21 (Fig. 2E,F), consistent with the previous study of Leon and colleagues (Leon et al., 2020;Carriere et al., 2020).cPcdhg ÀcKO mice developed normally to adulthood, and their body weights were also similar to the control and PV-cKO mice (Fig. 2G,H).

Deletion of cPcdhc in PV 1 cells does not affect the electrical membrane properties of PV 1 and Pyr cells
To evaluate the effect of cPcdhg deletion of on the membrane properties of PV 1 and Pyr cells in PV-cKO mice, whole-cell recordings were taken from PV 1 cells, which were visualized using tdTomato (Fig. 3A-K), and Pyr cells (Fig. 3L-U) in layer 2/3 (L2/3) of the primary visual cortex at P21-P26.There were no significant differences in the resting membrane potential, input resistance, and action potential kinetics of PV 1 cells and Pyr cells between control and PV-cKO mice.This suggests that cPcdhg deletion in PV 1 cells does not impact the electrical cell membrane properties.cPcdhc in PV 1 cells affect the dendritic morphology of PV 1 cells To identify the effect by PV-cKO on PV 1 cell morphology, dendrite morphologic analysis of biocytin-filled PV 1 cells was conducted (Fig. 4).We confirmed the cell type of the PV 1 cells by visualizing the morphology with biocytin injection and found no chandelier cells (Fig. 4A).Compared with control PV 1 cells, the complexity of dendrites within 50-150 mm from the soma was most significantly increased in PV-cKO PV 1 cells, and longer dendrites beyond 450 mm from the soma were significantly reduced in PV-cKO PV 1 cells (Fig. 4B,C).However, the number of primary dendrites, total dendritic length, number of branch points, and dendritic field area were similar between control and PV-cKO cells (Fig. 4D-G).

Deletion of cPcdhc in PV 1 cells on the excitatory does not significant affect translaminar and intralaminar inputs
Because the complexity and the length of dendrites of PV 1 cells were abnormal in PV-cKO mice (Fig. 4B,C), next we analyzed the neuronal connectivity between PV 1 cells and Pyr cells in the whole visual cortex.To determine whether cPcdhg deletion in L2/3 PV 1 cells causes any changes in local excitatory inputs onto PV 1 cells, we analyzed the laminar source and the strength of excitatory inputs to PV 1 cells using laser scanning photostimulation with caged glutamate (Yoshimura et al., 2005;Ishikawa et al., 2014).RuBi-glutamate was uncaged using blue light for the photostimulation of cortical cells from L1 to L6.To confirm that the action potential induction of cortical    neurons by photostimulation was comparable between the control and PV-cKO groups, loose patch-clamp recordings were made from L2/3 and L5 Pyr cells (Fig. 5A,  B).The photostimulation-evoked action potentials were observed only when the locations of the recorded cell were stimulated, and no action potentials were induced  at any other locations from L1 to L6 in both groups.The number of stimulation sites where action potentials were evoked by photostimulation in the recorded L2/3 and L5 Pyr cells (Fig. 5A), and the number of action potentials evoked by photostimulation at single stimulation sites in the recorded L2/3 and L5 Pyr cells (Fig. 5B), were not significantly different between the control and PV-cKO groups (Table 2).When photostimulation was applied near the PV 1 cell body, evoked EPSCs and direct responses were observed.Figure 5C shows that the direct response of PV 1 cells can be separated from photostimulation-evoked EPSCs inputs in temporal resolution (Dantzker and Callaway, 2000).Figure 5D presents representative spatial distributions of neurons presynaptic to the recorded PV 1 cells, with color-coding for the number and amplitude of photostimulation-evoked EPSCs.L2/3 PV 1 cells received excitatory inputs primarily from L2/3, L4, and moderately from L5 to L6 in both the control and PV-cKO groups (Fig. 5E,F), in accordance with previous reports (Dantzker and Callaway, 2000;Xu and Callaway, 2009).The cPcdhg -deleted PV 1 cells are no significant different from control mice in any layer (Fig. 5E,F).To verify more detailed excitatory input from L2/3 excitatory cells that are within the same layer, the input number and mean amplitudes were compared by distance from recording cell (Fig. 5H,I).As a result, no significant differences were found in PV-cKO groups (Table 2).
The deletion of cPcdhc in PV 1 cells increases the connection probability from PV 1 to 50-100 lm apart Pyr cells Because the complexity of dendrites within 50-149 mm from the soma was increased in PV-cKO PV 1 cells (Fig. 4C), next we performed additional simultaneous double whole-cell recordings from PV 1 cells and Pyr cells located 50-100 mm apart (Fig. 6).It was found that the percentages of PV 1 cells forming inhibitory synapses with Pyr cells were significantly increased in PV-cKO mice compared with control mice (Fig. 6B).However, the percentages of Pyr cells forming excitatory synapses onto PV 1 cells were not different between control and PV-cKO mice (Fig. 6C).There were also no differences in the probabilities of reciprocal connection of PV 1 cells and Pyr cells between control and PV-cKO mice  (Fig. 6A).In both control and PV-cKO mice, the amplitudes of uIPSCs of reciprocal pairs tended to be higher than those of one-way pairs (Fig. 6D), but there was no significant difference in uIPSC amplitude between control and PV-cKO mice (Fig. 6D).The amplitudes uEPSCs in the reciprocal pairs were also not significantly different between the control and PV-cKO mice (Fig. 6E).The deletion of cPcdhc in PV 1 cells does not affect the apparent connection probability or the properties of synaptic responses between Pyr and PV 1 cells below 50 lm Our previous findings showed that in the barrel cortex cell-lineage-dependent reciprocal connections between excitatory neuron pairs with intracellular distance below 50 mm are significantly reduced in cPcdh-deficient neurons Tarusawa et al., 2016).Next, we conducted simultaneous double whole-cell recordings from PV 1 cells and Pyr cells located within 50 mm of each other, to determine whether cPcdhg is involved in the connectivity between PV 1 cells and Pyr cells (Fig. 7A,B).In control mice, PV 1 cells established inhibitory synapses with Pyr cells in 91% of the recorded pairs.Pyr cells formed excitatory synapses onto PV 1 cells in 69% of cases, resulting in ;67% of pairs being reciprocally connected.The percentage of excitatory one-way connected pairs was only 2% (Fig. 7C).These findings align with previous research (Hofer et al., 2011;D'Souza et al., 2019).The connectivity in PV 1 cell specific PV-cKO mice was nearly identical to that in control mice (Fig. 7C).The impact of cPcdhg deletion on the strength of synaptic connections was assessed by analyzing the amplitude of the synaptic responses between the two cells.Previous research has shown that the amplitude of unitary IPSCs (uIPSCs) is significantly greater in reciprocal pairs than in inhibitory oneway pairs (Yoshimura and Callaway, 2005).We also found a significant difference in the amplitude of uIPSCs between reciprocal pairs and inhibitory one-way pairs (Fig. 7D), both in control and PV-cKO mice.However, no significant difference in uIPSC amplitude was found between control and PV-cKO mice (Fig. 7D).The amplitudes of unitary EPSCs (uEPSCs) in the reciprocal pairs were also not significantly different between the control and PV-cKO mice (Fig. 7K).These results indicate that the deletion of cPcdhg in PV 1 cells does not influence the connection probability between PV 1 cells and Pyr cells located within 50 mm of each other.Analysis of the waveform kinetics of the uIPSCs and uEPSCs showed that in both control and PV-cKO mice, the failure event rate, paired pulse ratio, half-width, rise time, and decay time of uIPSCs (Fig. 7E-J) and uEPSCs (Fig. 7L-Q) were similar.These results suggest that cPcdhg in PV 1 cells does not affect the properties of the synaptic responses at inhibitory and excitatory synapses.
Individual PV 1 cell-specific neural connectivity with Pyr cells was impaired by the deletion of cPcdhc in PV 1 cells Previous reports have demonstrated that PV 1 cells form synapses onto Pyr nonspecifically (Packer and Yuste, 2011).However, the converse is not true; Pyr cells can selectively target PV 1 cells (Yoshimura and Callaway, 2005).We further examined the connectivity of each PV 1 cell with multiple Pyr cells.We performed simultaneous whole-cell patch clamp recordings from a single PV 1 cell and two Pyr cells (Fig. 8A,B).In control mice, we found that reciprocally connected PV 1 cells received significantly more inputs from other Pyr cells compared with PV 1 cells connected in a one-way inhibitory fashion (Fig. 8B).These results indicate the two patterns of connectivity between PV 1 cells and Pyr cells: PV 1 cells that preferentially receive inputs from multiple Pyr cells, and PV 1 cells that receive fewer inputs from Pyr cells.Strikingly, this preference for excitatory inputs from Pyr cells disappeared in the cPcdhg -cKO mice.To quantify the bias in the inputs from the Pyr cells to individual PV 1 cells, we examined the connectivity between a single PV 1 cell and surrounding .3Pyr cells (Fig. 8C).The connectivity of each PV 1 cells with multiple Pyr cells was categorized.We calculated the connectivity of individual PV 1 cells as the reciprocity of PV 1 cells (RPV): the number of reciprocally connected pairs divided by the number of pairs that bind at inhibitory synapses (Fig. 8C).An RPV value of 1 indicates that all Pyr cells that receive inhibitory inputs from the PV 1 cell are connected reciprocally.We categorized PV 1 cells into three groups according to the RPV value: low-RPV (0 , RPV , 0.5), middle RPV (0.5 5 RPV , 1), and high-RPV (RPV ¼ 1).We found that 75% of PV 1 cells were categorized as having high-RPV in control mice, whereas the remaining PV 1 cells were equally distributed in the low or middle RPV (Fig. 8D) at P21-P26.In contrast, PV-cKO mice showed that middle RPV was most common (52%), and the proportion of high-RPV was reduced to 43% (Fig. 8D).PV 1 cells in the low-RPV group showed significantly lower amplitude of uIPSCs and higher paired pulse ratio and failure rate compared with PV 1 cells in the middle and high-RPV groups in control mice (Fig. 8F-H).Contrarily, PV 1 cells in the low-RPV group in PV-cKO mice, did not exhibit the characteristics of the low-RPV group in control mice.The amplitude of uIPSCs in the middle RPV in cKO mice was similar to that of high-RPV in control mice (Fig. 8F), indicating that high-RPV might become a middle RPV by the reduction of reciprocity in PV-cKO mice.Similar to those at P21-P26, different distributions of high-RPV, middle-RPV, and low-RPV between control and PV-cKO mice appeared at P35-P41 adult stages (Fig. 8E).These results suggest that cPcdhg determines the characteristics of excitatory synaptic partners in each PV 1 cell.

Discussion
We evaluated the effects of cPcdhg deletion in PV 1 cells on the formation of neural circuits in the visual cortex.We chose a developmental time point of cPcdhg deletion when peak programmed cell death has passed.Here, we found that connectivity of PV 1 -Pyr was significantly different between control and PV-cKO mice.Reciprocal connected PV 1 cells to Pyr cells were significantly higher than one way connected PV 1 cells in control mice.However, there were no significant differences in PV-cKO mice.The proportion of high reciprocal connected PV 1 cells to Pyr cells with large uIPSC amplitudes was reduced in PV-cKO mice, and the reciprocity of PV 1 cells connected to Pyr cells with large uIPSC amplitudes was reduced (Fig. 9).These results indicated that cPcdhg in PV 1 cells regulates their reciprocity with Pyr cells in the cortex.We demonstrated for the first time the function of cPcdhg in inhibitory neurons in neural circuit formation.
Influence of deletion of cPcdhc in PV 1 cells on the dendritic morphology of PV 1 cells Expression of cPcdhg is essential for dendritic formation in several neurons.Deletion of cPcdhg in Purkinje and starburst amacrine cells abrogates repulsion and causes self-recognition and increased self-crossing (Lefebvre et al., 2012).In excitatory neurons of layer 5 in the somatosensory cortex, dendritic complexity is reduced by cPcdhg deletion (Garrett et al., 2012;Molumby et al., 2016).Our results also revealed abnormal dendritic formation in PV 1 cells, evidenced by increased dendritic complexity between 100-149 mm from the soma and a reduction of longer dendrites because of cPcdhg deletion (Fig. 4).Therefore, it seems cPcdhg isoforms regulate dendrite formation in PV 1 cells as well.Influence of cPcdhc deletion in PV 1 cells on the cortical neural circuits Deletion of cPcdhg in inhibitory neurons during the early stages of cortical development caused neural cell death (Carriere et al., 2020;Leon et al., 2020).In particular, deletion of cPcdhg in inhibitory neurons leads to excessive cell death in the cortex from around P8 (Carriere et al., 2020).Programmed cell death of inhibitory cells is regulated by excitatory inputs from Pyr cells, indicating that cPcdhg may be involved in neural circuit formation (Wong et al., 2018).Here, using PV 1 -specific cPcdhg -deficient mice, we could examine the function of cPcdhg in neural circuit formation without causing cell death.Through laser-scan photostimulation of caged glutamate, no significant differences were observed in the excitatory input source and input strength of PV 1 cells in layer 2/3 between control and PV-cKO mice by Bonferroni's multiple comparisons test in Figure 5. Conversely, multiple whole-cell recordings of single PV 1 cells to multiple Pyr cells revealed two distinct types of PV 1 cells: high-RPV with large uIPSC, and middle-RPV and low-RPV with small uIPSC in 2/3 layer of the visual cortex in control mice.However, in PV-cKO mice, the proportion of high-RPV with large uIPSC decreased, and the proportion of middle-RPV with large uIPSC increased, suggesting that the reciprocity of PV 1 -Pyr connections is reduced in PV-cKO mice.This proportional abnormality in PV-cKO mice was initially observed at P21-P26 and was also observed at P35-P41 in the adult stage, suggesting that it is not caused by developmental delay in neural circuit maturation.In retinal starburst amacrine cells, cPcdhg is required for synapse elimination during development (Kostadinov and Sanes, 2015), indicating the possibility that the reduction of reciprocal connections between PV 1 cells and Pyr cells in PV-cKO mice may be because of impaired elimination of excitatory synapses during circuit development before P21.Interestingly, the ratio of reciprocal connectivity between clonal layer four excitatory cells in the barrel cortex increases from P9-P11 to P13-P16 in wildtype cells, but this increase does not occur in cPcdhdeficient cells.Additionally, further elimination of one-way connectivity is observed from P13-P16 to P18-P20 in wildtype cells, but this elimination does not occur in cPcdhdeficient cells (Tarusawa et al., 2016).Future analysis of PV 1 -Pyr connectivity early in life is needed to determine whether elimination is occurring.

Functional meaning of different reciprocal connectivity of PV 1 cells
As discussed above, we made a novel discovery that PV 1 cells can be categorized into different types in control mice: high-RPV with large uIPSC and low-RPV or middle-RPV with small uIPSC.While there are no reports on the proportion of reciprocal connections of PV 1 cells from multiple Pyr cells, it has been documented that several types of basket cells exist based on mRNA expression and cell morphology (Gouwens et al., 2019), and that each basket cell has different visual response characteristics (Hofer et al., 2011) and dendritic morphologies (Runyan and Sur, 2013).Recently, it has also been reported that certain PV 1 cells show different rates of reciprocal connections in other brain regions.Therefore, the functional and morphologic features of these two different types of PV 1 cells in relation to reciprocity and uIPSC amplitude observed in control mice need to be examined in future studies.
Elaborating functional significance of PV 1 cell types is beyond the scope of this study; however, two plausible hypotheses can be proposed.One hypothesis involves a preference for visual stimulus response.Excitatory cells in the primary visual cortex exhibit high orientation selectivity, responding only to visual stimuli of a specific orientation.However, only ;18% of PV 1 cells show a high degree of orientation selectivity, while most PV 1 cells are active in response to visual stimuli of any orientation (Hofer et al., 2011).This observation aligns with the results of our study, where 25% and 23% of low-RPV or middle-RPV cells were observed in P21-P26 and P35-P41, respectively, in control mice.Each Pyr cell has varying orientation selectivity.PV 1 cells that are reciprocally connected to many Pyr cells (high-RPV) can respond to multiple orientations and become less orientation-selective because of inputs from numerous Pyr cells.In contrast, PV 1 cells with reciprocal connections to only a few Pyr cells (low-RPV) may exhibit biased orientation selectivity.
The second hypothesis involves the thalamic input.Relay neurons in the lateral geniculate nucleus are mainly projected to L4 of the visual cortex, but there are also patchy projection areas in L1.In PV 1 -Pyr pairs in L2/ 3 under the patchy projection areas, the Pyr cells receive significantly smaller uIPSC amplitudes compared with the pairs under the interpatch, despite no difference in the connection probability of reciprocity (D 'Souza et al., 2019).We found that PV 1 cells within different types of local neural circuits significantly differ in terms of the rate of reciprocal connections and uIPSC amplitudes, suggesting a potential link between thalamic input and the location of PV 1 cells with high-RPV and low-RPV or middle-RPV characteristics.

Molecular mechanisms of regulation of neural circuit formation by cPcdhc
The increase of dendritic complexity near the PV 1 cell soma (50-200 mm) in PV-cKO mice may contribute to the increase in excitatory inputs, as the probability of PV 1 cell dendrites encountering axons of Pyr cells is increased.The probability of inhibitory synaptic connections within 50 mm of PV 1 cells was not affected by cPcdhg deletion; however, excitatory synapses were affected (Fig. 8), which aligns with the notion that PV 1 cells target adjacent Pyr cells nonspecifically (Packer and Yuste, 2011), while Pyr cells selectively target their binding partners (Yoshimura and Callaway, 2005).In this study, the probability of inhibitory synaptic connections from PV 1 cells located 50-100 mm away from Pyr cells was significantly increased in cPcdhg deletion (Fig. 6A), suggesting that cPcdhg may also regulate inhibitory synaptic connections from more distant PV 1 cells.Interestingly, the targeting probability from PV 1 cells to Pyr cells decreases with distance (Packer and Yuste, 2011).However, our results only examined the role of cPcdhg in PV 1 cells on PV 1 -Pyr connectivity.Since cPcdhg is also expressed in Pyr cells, it is important to analyze cPcdhg -cKO Pyr cells to further understand the molecular mechanisms of transinteractions of cPcdhg proteins in synaptic connectivity.

Figure 1 .
Figure 1.Confirmation of cPcdhg deficiency by lacking g CR1 exon.A, Genomic structure of the cPcdhg gene.The filled and open triangles represent loxP and frt sites, respectively.B, Gross phenotypes of P0 neonatal pups.cPcdhg DCR1/DCR1 mutants had a hunched posture in most cases and died within 1 d after birth.cPcdhg 1/DCR1 heterozygotes were survived.C, PCR genotyping used to distinguish between wild-type (cPcdhg 1/1 ) and cPcdhg DCR1/DCR1 mutants.D, Western blot analysis of the whole-brain lysate from P0 mouse brain.cPcdhg DCR1/DCR1 mutants did not express cPcdhg protein.b -Actin was used as the loading control.

Figure 5 .
Figure 5. PV 1 cells in PV-cKO mice receive same inputs from surrounding Pyr cells compared with control mice.A, The number of stimulus sites where cell firing was induced in recorded Pyr cells at L2/3 (left) and L5 (right) by laser photostimulation and their distance from the recording site is presented here.The bar indicates SEM.B, Number of spikes per laser photostimulation and their distance from the recording Pyr cells of L2/3 (left) and L5 (right), respectively.The bar indicates.C, Representative example of same EPSC traces by photostimulation.Bottom, Ten minutes after the addition of TTX (bottom).The blue bar represents the laser exposure period.D, Left, Image of a brain slice recorded from PV 1 cells in a layer 2/3 V1 region (right) and cytochrome c oxidase and Nissl-stained image of the same slice as in the right (left).Scale bar: 100 mm.Right, Photostimulation-evoked EPSCs (EPSCs) recorded in layer 2/3 PV 1 cells.Reconstructions of the locations of photostimulation sites (colored squares) relative to the locations of laminar borders and cell bodies of the recorded PV 1 cells (open black circles) are shown.The white squares indicate no input.Left, The color of each square indicates the sum of the amplitudes of EPSCs that were observed in response to photostimulation at that site.Right, The color of each square indicates the number of EPSCs events observed in response to photostimulation at that site.The EPSC traces of the photostimulation at each spot (indicated by a, b, c) are presented on the right.The blue bar represents the laser exposure period.E, The mean amplitude of photostimulation-evoked EPSCs for each layer is plotted.The bars indicate median 6 95% CI values.F, Mean number of events induced by photostimulation-evoked excitatory responses.The bars indicate median 6 95% CI values.G, L2/3 only input amplitude map (left) and input event map (right).The location of the recorded PV 1 cell is indicated by an open black circle and the number in the squares indicates the distance from the recording cell.H, Mean amplitude at each distance from the recorded PV 1 cells.The bar indicates SEM.I, Mean number of events at each distance from recorded PV 1 cells.The bar indicates SEM (A, B, H, I) Bonferroni's multiple comparisons test.Significance is indicated in the figures as follows: *p , 0.05, n.s.p .0.05.Refer toTable 2 for statistical information.

Figure 8 .
Figure 8. Deletion of cPcdhg in PV 1 cells affects the specificity of local neural connections between single PV 1 cell and multiple Pyr cells.A, Image of a brain slice with recording electrodes in the layer 2/3 V1 region (upper).Scale bar: 200 mm.Highmagnification images of triple whole-cell recordings (lower panel).Scale bar: 20 mm.B, Right, The Probability of excitatory inputs from a third party of Pyr cells on PV 1 cells with different connectivity.Left, Schematic illustration of three-cell connection

Figure 9 .
Figure 9. Effects of PV 1 cell-specific cPcdhg KO on neural circuit formation in mouse primary visual cortex.

Table 1 :
List of primer sequences

Table 2 :
Statistical information for the data shown in Figure5